C-Mannosylation is a recent type of protein glycosylation that involves the addition of a single mannose to WXXW acceptor sites in membrane and secreted proteins. Our preliminary data suggest the Cys subdomains in MUC5AC and MUC5B are C-mannosylated when expressed in cultured animal cells. Similarly to the critical function of the two other types of glycosylation in mucins N- and O-glycosylation, C-mannosylation might have an important role in mucin/mucus function. Indeed, the expression studies suggest C-mannoses are required for proper folding of the Cys subdomains, although a role in specific lectin-type interactions with mucus, bacteria, and/or epithelial proteins cannot be discarded. Consistent with these initial observations, the overal objective of this project is the structural and functional characterization of C-mannosylation in MUC5AC and MUC5B. In Specific Aim 1 we proposes to detect C-mannose in native MUC5AC/MUC5B. These mucins will be purified from primary cultures of human epithelial airway cells, digested with proteases and C-mannoses detected by mass spectrometry analysis and peptide sequencing of mucin peptides previously fractionated by reverse phase chromatography. Studies in Specific Aim 2 will test the hypothesis that C-mannosylation is required for proper folding of the mucin Cys subdomains. Thus, these domains will be expressed in and purified from C-mannosylation-deficient and normal cells, respectively, and their overall structures assess by peptide mapping and analysis of their respective CD spectras. Together, these studies should provide solid data to discern whether or not the role of the Cys subdomains in mucins, a protein domain that shows the highest degree of protein conservancy among human mucins, is linked to the presence of C-mannose.